For the structural analysis of the catalytic domain of Golgi glycosyltransferases, in particular beta-1,4-galactosyltransferase (beta1,4-GT) that transfers galactose from UDP-galactose to N- acetylglucosamine (NAG), either free or bound to an oligosaccharide, a number of N- and C-terminal deletion and point mutants of the enzyme were expressed in E. coli to determine the binding regions of the enzyme that interact with the acceptor or donor substrates. The N-terminal truncated forms of the enzyme between the residues 1-129, do not show any difference in the apparent Km's towards NAG or linear oligosaccharide acceptors e.g., for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, GT-129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, GT- 129NAG, that contains the residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active GT-129. Unlike the C-terminus fragment GT-257UDP, containing residues 258-402, the N- terminus fragment, GT-129NAG, that contains the residues 130-257 of the beta-1,4GT, binds less efficiently to UDP-columns and can be eluted from the column with only 15 mM NAG. On the otherhand, the C- terminus fragment, GT-257UDP, binds tightly to both NAG- and UDP- agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behavior of N- and C-terminal fragments of beta- 1,4GT towards the NAG- and UDP-agarose columns differ, the former binding preferentially to NAG-columns, while the latter binds to UDP- agarose columns via Mn+2.